Background

The increasing levels of Plasmodium falciparum resistance to chloroquine (CQ) in Thailand have led to the use of alternative antimalarials, which are at present also becoming ineffective. In this context, any strategies that help improve the surveillance of drug resistance, become crucial in overcoming the problem.

Methods

In the present study, we have established the in vitro sensitivity to CQ, mefloquine (MF), quinine (QUIN) and amodiaquine (AMQ) of 52 P. falciparum isolates collected in Thailand, and assessed the prevalence of four putative genetic polymorphisms of drug resistance, pfcrt K76T, pfmdr1 N86Y, pfmdr1 D1042N and pfmdr1 Y1246D, by PCR-RFLP.

Results

The percentage of isolates resistant to CQ, MF, and AMQ was 96% (50/52), 62% (32/52), and 58% (18/31), respectively, while all parasites were found to be sensitive to QUIN. In addition, 41 (79%) of the isolates assayed were resistant simultaneously to more than one drug; 25 to CQ and MF, 9 to CQ and AMQ, and 7 to all three drugs, CQ, MF and AMQ. There were two significant associations between drug sensitivity and presence of particular molecular markers, i) CQ resistance / pfcrt 76T (P = 0.001), and ii) MF resistance / pfmdr1 86N (P < 0.001)

Conclusions

i) In Thailand, the high levels of CQ pressure have led to strong selection of the pfcrt 76T polymorphism and ii) pfmdr1 86N appears to be a good predictor of in vitro MF resistance.

Malaria due to Plasmodium falciparum affects 300 million people and claims an estimated 1.5 million lives every year. Our present inability to synthesise a fully protective vaccine means that chemotherapy stands as the only effective measure in the control of the disease. However, in many parts of the world the parasite P. falciparum has become resistant to most drugs presently used [1], seriously undermining efforts for controlling malaria.

Chloroquine (CQ) has long been the drug of choice for the treatment of malaria; however, CQ-resistant parasites are now present in most areas where malaria is endemic [2]. Chloroquine resistance is especially well established in Thailand, after having been first described in that country in the late 1950's [3]. The decline in the efficacy of chloroquine has led to the use of alternative antimalarials, such as antifolates, mefloquine and artemisinin derivatives, but parasite resistance to these drugs is also becoming a real problem [2]. In this context, understanding the genetic basis of drug resistance is essential for implementing rational measures to overcome the problem.

Although significant progress has been made in trying to understand how resistance to CQ may occur, many aspects of it remain unclear, and the genetic mechanisms responsible for mefloquine and quinine resistance are largely unknown. Nevertheless, two main genes have been implicated in quinoline resistance; the pfmdr1 (P. falciparum multi-drug resistance1) and the Pfcrt (P. falciparum chloroquine resistance transporter). There is evidence from the analysis of a genetic cross which indicates that point polymorphisms in the pfmdr1 gene may modulate sensitivity to both mefloquine (MF) and artemisinin in P. falciparum[4]. Furthermore, recent genetic transfection work has suggested that single nucleotide polymorphisms in the pfmdr1 gene encoding changes in aminoacids 1034, 1042 and 1246 can influence parasite responses to mefloquine, quinine and halofantrine as well as to the structurally unrelated drug artemisinin, and modulate sensitivity to chloroquine depending on the genetic background of the parasites strains [5]. However, chloroquine resistance was shown to segregate independently of the pfmdr1 gene, following a genetic cross between a CQ-sensitive parasite, P. falciparum HB3, and a CQ-resistant one, Dd2 [6], and the absence of a clear association between pfmdr1 and chloroquine responses in natural parasite populations [7–15], strongly suggests the involvement of other gene(s). Recently, detailed linkage analysis and fine chromosome mapping of progeny clones of the HB3 × Dd2 cross has allowed the identification of another gene, pfcrt, in which a mutation at aminoacid 76 (pfcrt K76T) is highly correlated with increased CQ tolerance among field parasite isolates of P. falciparum[16–24]. In addition, a causal relationship between pfcrt 76T and chloroquine resistance has been confirmed by genetic transfection experiments [16].

The study of the correlation between drug resistance in natural parasite populations and genetic polymorphisms may allow the development of molecular tools to help predict responses to drugs and, as mentioned above, the pfcrt and pfmdr1 genes have been identified as putative markers of quinoline resistance. In the present work we have investigated possible associations between four molecular markers in these genes and sensitivity to chloroquine, mefloquine, quinine (QUIN) and amodiaquine (AMQ) of P. falciparum parasites collected in Thailand.

Study site and method of parasite collection

After confirmation of P. falciparum infection by microscopical observation of thin and thick Giemsa-stained blood films, approximately 20 μl of blood were collected by finger-prick from consenting subjects living in 4 different areas of Thailand where malaria is highly endemic; the Tak province (North-western Thailand), Kanchanaburi (Western Thailand: Thai-Myanmar border), Chonburi (Eastern Thailand), Chantaburi and Trat (Eastern Thailand: Thai-Cambodia border). Samples were placed in sterile 1.5 ml micro-centrifuge tubes, together with 0.5 ml of transport medium (10 ml RPMI 1640 complete medium without serum, plus 20 μl of heparin 5000 i.u./ml), carried to Chulalongkorn University of Bangkok on the day of collection, at ambient temperature and placed in in vitro culture, following established procedures [25].

Parasite phenotyping (micro-tests)

Assessment of P. falciparum susceptibility to CQ, MF, QUIN and AMQ was performed according to the M.I.C. test [26, 27], whereby samples were exposed to a range of concentrations of the four drugs, in microtitre culture plates, for 72 hours, with daily changes of medium (in the presence or absence of drugs). After this period, thin film Giemsa-stained preparations from each culture plate well were observed under light microscopy for the presence of parasites. The results were expressed in "minimum inhibiting concentration" (M.I.C.) units, i.e., the lowest drug concentration required to kill all or nearly all parasites (I.C.99) after 72 hours [26, 27].

Parasite reference strains of known sensitivity to the drugs concerned were tested in parallel to all field-collected isolates. In this manner, the drug response of each isolate was determined by comparison of the M.I.C. between each sample and the relevant reference strains. Accordingly, samples tested for CQ, MF, QUIN and AMQ responses were clustered into two groups; sensitive (S) or resistant (R) based on sensitivity thresholds established in previous studies [26–29].

Genotyping by PCR-RFLP

Genomic DNA was extracted prior to drug testing, by a standard phenol:chloroform DNA extraction method, precipitated with absolute ethanol and stored in TE Buffer (Tris-EDTA), following established protocols [30]. The resulting DNA was used as template in 50 μl PCR reactions, containing 1 μM of each oligonucleotide primer, 1 × PCR buffer (Promega™), 2.5 mM MgCl2, 0.2 mM dNTP's and 0.025 U/μl of Promega™ Taq DNA polymerase. Accordingly, a fragment of the pfcrt gene containing codon 76 was amplified by PCR using a Nested-PCR approach. For amplification of DNA fragments containing pfmdr1 polymorphisms we used oligonucleotide primers published elsewhere [31], as well as newly designed ones. In this manner, the fragments of the pfmdr1 gene containing codons 86 and 1246 were amplified in a single-step PCR, whereas the sequence of codon pfmdr1 1042 was determined following amplification by semi-nested PCR. All primer sequences and respective PCR conditions are presented in Table 1.

Restriction enzymes generating RFLPs

Following amplification of the fragments concerned, polymorphisms in the pfcrt and pfmdr1 genes were assessed as follows: pfcrt 76K and pfmdr1 86N were detected by incubation of the corresponding PCR fragments with Apo I (r/aatty), pfmdr1 1042N was detected using Asn I (at/taat), and pfmdr1 1246Y was determined by incubation with Eco RV (gat/atc). Endonucleases Apo I, Asn I and Eco RV had been obtained from New England BioLabs™, Roche Molecular Biochemicals™ and Stratagene™ respectively, and incubations were setup following the manufacturers instructions. Appropriate control DNA of samples with known pfcrt and pfmdr1 sequences was used in parallel with field-collected parasite isolates in every PCR-RFLP protocol; these were 3D7 (genotype pfcrt 76K, pfmdr1 86N, pfmdr1 1042N, pfmdr1 1246D), HB3 (genotype pfmdr1 1042D), Dd2 (genotype pfcrt 76T, pfmdr1 86Y) and 180/92 (genotype pfmdr1 1246Y). The products resulting from restrictions of pfmdr1 1042 were resolved in 8% acrylamide gels, whereas pfmdr1 86, pfmdr1 1246 and pfcrt 76 digests were run on 2% agarose gels, with both types of gels made in 1 × TBE buffer. All gels were stained with ethidium bromide and visualised under UV (ultraviolet) transillumination.

Statistical analysis of the association between pfcrt and pfmdr1 markers and drug responses

We searched for statistically significant associations between sensitivity to each of the drugs among all isolates and the presence of each of the particular markers included in our study by using Fisher's Exact Test (2-tailed) after having arranged the data in 2 × 2 contingency tables (mixed infections were excluded from this analysis). An association between a particular marker and resistance to a given drug was considered to be significant if the P value was found to be lower than 0.05 (P < 0.05).

Pfcrt and pfmdr1 genotypes

The pfcrt 76 PCR primers amplified a sub-fragment of the gene comprising 479 nucleotides, containing both a monomorphic and a polymorphic Apo I restriction site. In this manner, incubation of this fragment with endonuclease Apo I generated either 3 fragments of 122, 124 and 233 base-pairs (b.p.), or 2 fragments of 233 and 246 b.p., if K or T were present respectively (Figure 1). The PCR of pfmdr1 86 amplified a fragment of 504 b.p., within which the presence of asparagine (N) was determined by incubation with Apo I, originating segments of 255 and 249 b.p., which resolved as a single band in 2% agarose gels (Figure 1). For detection of 1042 polymorphisms (N or D) the 188 b.p. fragment obtained by PCR could be restricted with endonuclease Asn I, after which 3 segments were obtained of 116, 46 and 26 b.p. if 1042N were present in the sample, and two fragments of 162 and 26 b.p. in the presence of the alternative aminoacid (as with pfcrt 76, one of the restriction sites for Asn I within this segment is also monomorphic, therefore always resulting in at least one cut) (Figure 1). Codon 1246 polymorphisms (D or Y) were assayed following incubation of the corresponding 508 b.p. PCR product with Eco RV, which produced 2 fragments of 268 and 240 b.p. if the target DNA contained tyrosine (Y) (Figure 1). All data resulting from this analysis is compiled in Table 3, where allele frequencies for each of the markers are shown.

Association between pfcrt and pfmdr1 markers and responses to drugs

Two significant correlations were detected between the presence of a particular marker and in vitro outcomes (Tables 4 and 5); one between CQ resistance and the presence of pfcrt 76T (P = 0.001) and the other, between MF resistance and the presence of pfmdr1 86N (P < 0.001). There was an evident lack of an association between AMQ responses and all of the markers studied (Table 6), a fact that was corroborated after statistical analysis (data not shown). In addition, since all isolates proved to be quinine sensitive, any correlations between response to this drug and corresponding genotypes could not be established. The data of the in vitro tests for the 4 drugs, and corresponding genotypes is compiled in Table 7.

Table 4

Plasmodium falciparum isolates listed according to sensitivity to chloroquine (CQ), with corresponding pfcrt and pfmdr1 markers

The increasing failure rates of several antimalarial drugs in the majority of malaria-affected areas means that close monitoring of the epidemiology and dynamics of drug resistance are necessary if we are to implement measures to circumvent the problem. The identification and validation of easy, rapid molecular markers of drug resistance would greatly facilitate this process, and would allow us to overcome difficulties in the use of traditional methods for assaying drug sensitivity.

In Thailand, CQ resistance was first reported more than forty years ago [3], and after ten years, resistance to chloroquine had become so widespread that use of the drug against P. falciparum was abandoned. At present, even though the drug is used only against P. vivax, it is perhaps not surprising to find that most P. falciparum are largely unaffected by CQ. Our in vitro observations show a near total prevalence of CQR (96%) in the present study area, confirming what has been widely reported [1]. Among all CQR isolates tested in our study, the presence of pfcrt 76T was universal (P = 0.001), indicating complete selection of this polymorphism by the drug, a result that is in agreement with recently published work not only from Thai-originated parasites [22], but also from parasites of different areas of the globe [32].

The correlation between pfmdr1 genotypes and quinoline resistance has often generated conflicting results; although it has been suggested that pfmdr1 86Y can be correlated with increased CQ resistance in parasites which originated from different areas of the world [33–36], other field studies have not corroborated these findings [7–15], and the results of a P. falciparum genetic cross indicated that CQR did not depend on the pfmdr1 gene [6]. Our present findings do not implicate pfmdr1 86 in CQ resistance in Thailand, since the presence of both N and Y in our samples was largely independent of their CQ response, indicating that chloroquine does not appear to exert selective pressure on this area of the gene. Interestingly though, pfmdr1 1042D and pfmdr1 1246N, previously associated with increased CQ sensitivity following genetic transfection experiments [5], were largely absent in our samples, possibly suggesting a mechanism of chloroquine resistance that may in part depend on the presence of the alternative polymorphic alleles, pfmdr1 1042N and pfmdr1 1246D, respectively.

Mefloquine was introduced in Thailand in the form of Fansimef (mefloquine-sulfadoxine-pyrimethamine) during 1984 with an initial cure rate of 95%, but MF-resistant P. falciparum parasites have arisen and present a real threat to the control of malaria, especially in the Thai/Cambodia and Thai/Myanmar border areas [37]. In the present work the prevalence of MF resistance was 62%, indicating a worrying trend. A correlation between pfcrt 76T and mefloquine sensitivity would always be difficult to establish, since the near complete presence of this polymorphism is likely to have been selected by chloroquine pressure, whose mechanisms of action and resistance are probably distinct from those of MF. Earlier work from Thailand and other areas of the world has indicated that an increase in the level of mefloquine sensitivity among field isolates of P. falciparum may be correlated with a mutation in codon 86 of the pfmdr1 gene (N86Y) [38–40]. In the present work, the occurrence of pfmdr1 86N was significantly associated with MF resistance as 31/32 resistant isolates carried this polymorphism (P < 0.001), strongly suggesting that 86N is an important event in the generation of MF resistance and may be a useful marker to monitor MF resistance in this country.

Interestingly, although the prevalence of AMQ resistance was high (58%), we did not detect a significant correlation between AMQ responses and any of the markers studied, contrary to what could be expected considering that AMQ is chemically very similar to CQ. These observations indicate that the mechanism of action and/or resistance differ between the two drugs, which may raise interesting questions about the design of new CQ-derivative compounds.

Quinine is one of the most effective drugs for the treatment of malaria in Thailand. Although the efficacy of this drug has been reduced, it has not yet become a serious problem, since the combination with tetracycline increases its cure rate. In fact, P. falciparum quinine sensitivity was total in all regions covered by our study, showing that despite its adverse side effects, quinine can still be used as a reliable resource of malaria therapy in Thailand. The reasons for the long-lasting efficacy of quinine may be manyfold, but its explanations lie outside the scope of this article. Most importantly, quinine may prove especially valuable in the treatment of multi-drug resistant Falciparum malaria, which we found to be largely present in our study as 41 out of 52 isolates were found to be resistant to more than one compound and 7 were unaffected by all drugs (CQ, MF and AMQ) except quinine.

Taken together, our results seem to be suggesting that CQ and MF are the major selective forces on the pfcrt and pfmdr1 genes, whereby the presence of pfcrt 76T, and possibly pfmdr1 1042N and pfmdr1 1246D in Thai-originated parasites has been selected by chloroquine pressure. The pfmdr1 86N mutation seems to be important only for mefloquine resistance, and may represent a useful marker for monitoring resistance in this country, although its validation may require in vivo correlates and the analysis of a larger number of samples.

Acknowledgements

Dinora Lopes, Fátima Nogueira, José Pedro Gil, Virgilio do Rosário and Pedro Cravo were funded by the Ministério da Ciência e Tecnologia, Portugal. This work was partially supported by Fundação para a Ciência e Tecnologia (FCT), project PRAXIS XXI P/SAU/14070/98. Dinora Lopes and Pedro Cravo are grateful to David Walliker, Paul Hunt and Hamza Babiker from the Institute of Cell, Animal and Population Biology, University of Edinburgh, for excellent scientific advice and support. The work in Thailand was supported by malaria grants from Institute of Health Research, Faculty of Science, Chulalongkorn University. We thank the Malaria Division, Ministry of Health (Bangkok) and especially the staff of the Malaria Clinics for their co-operation.

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Authors' contributions

DL carried out a proportion of the parasite phenotyping, performed the molecular analysis of the majority of the samples and contributed for the elaboration of the manuscript. KR performed the molecular typing of a number of the isolates. FN participated in the parasite's phenotyping and genotyping. AS performed parasite collection and did a proportion of the micro-tests. JPG, VR and PC, conceived the study, participated in its design and co-ordination and were involved in phases of the experimental work.

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